MAMMALIAN COG4

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 高尔基体在真核细胞分泌途径中的蛋白质分选和糖解中起着关键作用。高尔基体内囊泡运输的缺陷影响其结构和功能。因此，这种缺陷可能会对细胞表面蛋白的糖基化和稳定性产生多效性影响，从而导致人类疾病。 这项建议源于美国国立卫生研究院资助的工作(RO1GM071574：“高尔基体系留蛋白的结构分析”)。总的来说，我们在这方面的努力主要集中在两个大的复合体上，即保守的寡聚体高尔基(COG)复合体和DSL1复合体。两者都是细胞内转运所必需的，并被认为在细胞内转运囊泡与其膜靶标的最初捆绑中起作用。然而，尽管进行了大量的努力，但人们对这些复合体在囊泡拴系中发挥作用的分子机制知之甚少。我们的长期目标是确定COG(8个亚基)和DSL1(4个亚基)络合物的亚基和亚组分的晶体结构。我们已经做了广泛的前期工作，特别是与COG，在表征综合体的整体架构(即亚单元连接性)方面。然而，这两个复合体的任何部分都没有公开的结构。我们最近已经获得了每个络合物的亚基的有希望的晶体，并建议在NSLS上使用MAD位相来确定它们的结构。 在这里，我们请求X-25或X-29波束时间来确定哺乳动物Cog4片段的结构。我们称其为265个残基的Cog4B。使用我们实验室的R-Axis IV图像板探测器，我们目前的Cog4B晶体可以很好地衍射X射线，至少达到3.0埃。因此，目前的努力集中在获得SeMet取代的晶体以用于MAD数据收集。由于对于这种大小的蛋白质来说，甲硫氨酸残基的总数很低(265个残基上有4个甲硫氨酸)，高质量的同步加速器数据很可能有助于MAD阶段的确定。鉴于科学的重要性和初步数据的前景，我们认为尽快收集数据是至关重要的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Golgi apparatus plays a key role in protein sorting and glycoslyation within the eukaryotic secretory pathway. Defects in vesicular trafficking within the Golgi affect both its structure and function. As a consequence, such defects can have pleiotropic effects on the glycosylation and stability of cell surface proteins, leading to human disease. This proposal arises from work funded by the NIH (RO1 GM071574: "Structural Analysis of Golgi Tethering Proteins"). Our efforts in this area, generally speaking, are focused on two large complexes, the Conserved Oligomeric Golgi (COG) complex and the Dsl1 complex. Both are essential for intracellular trafficking and are thought to act in the initial tethering of intracellular trafficking vesicles to their membrane targets. Nonetheless, despite intensive efforts, little is known about the molecular mechanism by which these complexes function in vesicle tethering. Our long-term goal is to determine crystal structures of subunits and subassemblies of the COG (8 subunits) and Dsl1 (4 subunits) complexes. We have done extensive preliminary work, especially with COG, in characterizing the overall architecture (i.e. subunit connectivity) of the complex. There are, however, no published structures of any portion of either complex. We have recently obtained promising crystals of subunits of each complex, and propose to determine their structures at NSLS using MAD phasing. Here, we write to request X-25 or X-29 beamtime to determine the structure of a fragment of mammalian Cog4. We call this 265-residue fragment Cog4B. Our current Cog4B crystals diffract x-rays well, to at least 3.0 angstroms using our laboratory's R-Axis IV image plate detector. Therefore, current efforts are focused on obtaining SeMet-substituted crystals for MAD data collection. Since the total number of Met residues is low for a protein of this size (4 Met over 265 residues), high-quality synchrotron data is likely to be instrumental for MAD phasing. Given the importance of the science and the promise of the preliminary data, we feel it is vital to collect data as soon as possible.